Septic (endotoxic) shock is advancing in rank as a significant cause of mortality. We will test the hypothesis that bacterial endotoxins act at the microcirculation to induce long-term alterations in microvascular endothelial cell (MEC) arachidonic acid (AA) metabolism. The resultant enhanced rate of basal prostaglandin E2 production may be responsible for the immunodepression and altered cardiovascular physiology observed in the septic shock state. The primary aim of this proposal is to fully characterize the mechanism by which endotoxins interact with MECs to initiate de novo AA metabolism. The model to be used is a primary culture of continuous, microvascular endothelial cells derived from rabbit or human adipose tissue. Initial studies will characterize the time course and product formation profile of AA metabolism in MECs in response to endotoxin. Dose-response relationships will be established for eicosanoid formation initiated by endotoxins derived from several species of bacteria. The binding of radio-labeled endotoxin to intact MECS and to isolated plasma membrane fragments will be analyzed by Scatchard analysis to calculate endotoxin receptor number and affinity. Electrophoretic techniques will be used to isolate and characterize endotoxin receptors present in MECs. Competition studies using biologically inactive endotoxins will be made to further characterize the endotoxin interaction with the cell membrane. Additional studies will identify the subcellular mechanism by which endotoxins initiate eicosanoid biosynthesis. Studies will be carried out using Western and Northern blot analysis to determine whether bacterial endotoxins affect the transcriptional or translational regulation of prostaglandin endoperoxide synthase, the key enzyme responsible for eicosanoid synthesis. Experiments will be done to irreversibly inhibit prostaglandin endoperoxide synthase and monitor the rate of return of enzymatic activity as an index of genomic expression of the enzyme. Other studies will determine the role of phospholipase A2, an enzyme that releases AA from membrane phospholipids, and the role of fatty acid deacylation/reacylation enzymes in providing substrate for the eicosanoid system. A final group of studies will investigate the possible second messenger system(s) that link the interaction of endotoxins at the plasma membrane to the mechanisms by which the arachidonic acid cascade is activated. These systems include protein kinase C, guanine nucleotide binding proteins, the phosphatidylinositol cycle, tyrosine kinase, and cyclic nucleotides. In summary, these studies will fully characterize the interaction between bacterial endotoxins and MECs that results in enhance, long-term eicosanoid production. It is suggested that this altered eicosanoid production plays a significant role in the altered microvascular and immunologic responses to endotoxic shock. A knowledge of the mechanisms by which endotoxin alters endothelial cell function is important to the design of effective therapeutic approaches to endotoxin- associated disease states such as pediatric and geriatric septic shock, burn-associated septicemia, surgical sepsis and septicemia associated with the acquired immune deficiency syndrome.